ABSTRACT This application addresses a significant gap in neurotoxicology, i.e. the role of gut microbiome. The microbiome of the human intestinal tract has a profound effect on human health through its key role in a wide range of host- related functions. Mounting evidence indicates that dysregulated gut microflora contribute significantly to a variety of human diseases. The fact that the gut microbiome can be readily affected by external factors raises questions regarding the role of xenobiotics on intestinal microflora. Glyphosate-based herbicides, such as Roundup, are the most widely used pesticides worldwide. Glyphosate acts on the shikimate pathway in plants through inhibiting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which disrupts the synthesis of aromatic amino acids, leading to plant death. Since the shikimate pathway does not exist in mammals, it is generally believed that glyphosate would be safe in humans. However, glyphosate adverse effects, including neurotoxicity, in mammals has been well documented. Of particular importance, many functionally important gut bacteria of rodents and humans do have the shikimate pathway, highlighting that glyphosate may perturb the gut microbiome and associated shikimate pathway to alter the homeostasis of aromatic amino acids, precursors of monoamine neurotransmitters. Of note, neurobehavioral disorders are often characterized with dysregulated aromatic amino acids and neurotransmitter pathways. However, the functional interaction between the gut microbiome and glyphosate exposure, especially at doses relevant to human exposure, is largely unexplored yet. The objective of this particular application is to define the impact of glyphosate on disturbing the gut microbiome and the role of glyphosate-disrupted microbiome in provoking neurotoxicity in the host. We will approach the problems in three stages by first characterizing the changes in the gut microbiome profiles with 16S rRNA sequencing, and then using shotgun metagenomics and metabolomics to map metabolic alterations. Lastly, we will define how glyphosate-perturbed gut microbiome causatively alters metabolome, shikimate and relevant pathways, neurotransmitters and neurobehavioral phenotypes via microbiome transplantation. Our proposed study is significant and represents a new frontier in glyphosate research because we focus on gut microbiome perturbation as a novel mechanism of its neurotoxicity. At the completion of this project, it is our expectation that these results will lay a foundation for future studies aiming at expanding our understanding of glyphosate neurotoxicity and the role of gut microbiome in human diseases caused by exposure to glyphosate, the most widely and heavily used herbicide in history.